Introduction:

The prostate gland may be a source of many health problems in men
past middle age, the most common benign prostatic hyperplasia
(BPH), and prostatic carcinoma (PCa). BPH is a noncancerous
enlargement of the prostate gland leading to obstruction of the
urethra and can significantly impair quality of life. The prevalence
of histological BPH is found in approximately 50-60% of males
age 40-50 and greater than 90% of men over 70 years old [1, 2]. In
many Western industrialized countries, including North America,
PCa is the most frequently diagnosed form of noncutaneous
malignancy in males. Except for lung cancer, PCa is the leading
cause of death from cancer [3-8]. Although the etiology of BPH
and PCa is unknown, some trace elements have been highlighted
in the literature in relation to the development of these prostate
diseases [9-29].

Trace elements have essential physiological functions such
as maintenance and regulation of cell function and signalling,
gene regulation, activation or inhibition of enzymatic reactions,
neurotransmission, and regulation of membrane function. Essential
or toxic (mutagenic, carcinogenic) properties of trace elements
depend on tissue-specific need or tolerance, respectively [30].
Excessive accumulation, deficiency or an imbalance of the trace
elements may disturb the cell functions and may result in cellular
degeneration, death and malignant transformation [30].

In earlier reported studies [31-69] significant changes of trace
element contents in hyperplastic and cancerous prostate in
comparison with those in the normal prostatic tissue were
observed. In particular, it was shown that the average mass
fraction of some trace elements in BPH were higher than normal
levels, while those in adenocarcinoma were lower than in healthy
prostatic parenchyma [60,61,66, 67,68]. Obtained results formed
the basis for a new method for differential diagnosis of BPH and
PCa, the essence of which was to determine the ratios of chemical
element contents changed in opposite directions during malignant
transformation of prostate. For example, a significant informative value of Zn/Co content as a tumor marker for PCa diagnostics was
shown by us [70]. Hence it is possible that besides Zn, the ratio of
Co to other trace elements also can be used as tumor markers for distinguish between benign and malignant prostate.

Currently number of methods was applied for the measurement of
chemical elements contents in samples of human tissue. Among
these methods, the instrumental neutron activation analysis with
high resolution spectrometry of long-lived radionuclides (INAALLR)
is a non-destructive and one of the most sensitive techniques.
It allows measure the chemical element contents in few milligrams
tissue without any treatment of sample. Nondestructive method of
analysis avoids the possibility of changing the content of trace
elements in the studied samples [71-74], which allowed for the
first time to obtain reliable results. However, INAA-LLR allows
only determine the mean mass fractions of 10-11 trace elements
in the samples of normal and cancerous prostate glands [15,28].
The inductively coupled plasma mass spectrometry (ICP-MS)
is more power analytical tool than INAA-LLR [18], but sample
digestion is a critical step in elemental analysis by this method. In
the present study two these analytical methods were used and the
results, obtained for some trace elements by ICP-MS, were under
the control of INAA-LLR data.

The present study had three aims. The main objective was to obtain
reliable results about the 43 trace elements: Ag, Al, Au, B, Be, Bi,
Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Mo,
Nb, Nd, Ni, Pb, Pr, Rb, Sb, Sc, Se, Sm, Sn, Tb, Th, Ti, Tl, Tm, U, Y,
Yb, Zn, and Zr contents in intact prostate of healthy men aged over
40 years and also in the prostate gland of age-matched patients,
who had either BPH or PCa, combining in consecutive order nondestructive
INAA-LLR with destructive ICP-MS method. The
second aim was to calculate Co/trace element content ratios for
every samples and compare the levels of these ratios in normal,
hyperplastic, and cancerous prostate. The third and final aim was
to evaluate the ratios of Co/trace element contents for diagnosis of
prostate cancer.
All studies were approved by the Ethical Committees of the
Medical Radiological Research Centre, Obninsk.

Material and Methods
Samples

The patients studied (n=92) were hospitalized in the Urological
Department of the Medical Radiological Research Centre
(Obninsk, Russia). All of them were European-Caucasian, citizens
of Moscow and Obninsk (a small city in a non-industrial region
105 km south-west of Moscow). Transrectal puncture biopsy of
suspicious indurated regions of the prostate was performed for
every patient, to permit morphological study of prostatic tissue
at these sites and to estimate their chemical element contents.
In all cases the diagnosis has been confirmed by clinical and
morphological results obtained during studies of biopsy and
resected materials. The age of 32 patients with BPH ranged from
56 to 78 years, the mean being 666 (MSD) years. The 60
patients aged 40-79 suffered from PCa (stage T1-T4). Their mean
age was 6510 (MSD) years.

Intact prostates (N) were removed at necropsy from 37 men aged
41-87 who had died suddenly. All deceased were EuropeanCaucasian,
citizens of Moscow. Their mean age was 5511
(MSD) years. The majority of deaths were due to trauma. Tissue
samples were collected from the peripheral zone of dorsal and
lateral lobes of their prostates, within 2 days of death and then
the samples were divided into two portions. One was used for
morphological study while the other was intended for chemical
element analysis. A histological examination was used to control
the age norm conformity, as well as to confirm the absence of
microadenomatosis and latent cancer [14,15,20,28].

Sample preparation

All tissue samples were divided into two portions. One was used
for morphological study while the other was intended for trace
element analysis. After the samples intended for trace element
analysis were weighed, they were freeze-dried and homogenized.
The sample weighing about 10 mg (for biopsy materials) and
50-100 mg (for resected materials) was used for trace element
measurement by INAA-LLR. The samples for INAA-LLR were
wrapped separately in a high-purity aluminum foil washed with
double rectified alcohol beforehand and placed in a nitric acidwashed
quartz ampoule.

After INAA-LLR investigation, the prostate samples were taken
out and used for ICP-MS method. The samples were decomposed
in autoclaves; 1.5 mL of concentrated HNO3 (nitric acid at 65 %,
maximum (max) of 0.0000005 % Hg; GR, ISO, Merck) and 0.3 mL
of H2O2 (pure for analysis) were added to prostate tissue samples,
placed in one-chamber autoclaves (Ancon-AT2, Ltd., Russia) and
then heated for 3 h at 160–200 °C. After autoclaving, they were
cooled to room temperature and solutions from the decomposed
samples were diluted with deionized water (up to 20 mL) and
transferred to the plastic measuring bottles. Simultaneously,
the same procedure was performed in autoclaves without tissue
samples (only HNO3+H2O2+ deionized water), and the resultant
solutions were used as control samples.

Instrumentation and methods

A vertical channel of a nuclear reactor was applied to determine
the trace element mass fractions by INAA-LLR. The quartz
ampoule with prostate samples and certified reference materials
was soldered, positioned in a transport aluminum container, and
exposed to a 24-hour neutron irradiation in a vertical channel
with a neutron flux of 1.31013 ncm-2s-1. Ten days after
irradiation samples were reweighed and repacked. The samples
were measured for period from 10 to 30 days after irradiation. The
duration of measurements was from 20 min to 10 hours subject to
pulse counting rate. The gamma spectrometer used for INAA-LLR
included the 100 cm3 Ge(Li) detector and on-line computer-based
multichannel analyzer. The spectrometer provided a resolution of
1.9 keV on the 60Co 1332 keV line. Other details of the INAALLR
analysis were presented in our previous publication [15].

An ICP-MS Thermo-Fisher “X-7” Spectrometer (Thermo Electron, USA) was used to determine the content of trace elements by
ICP-MS. The element concentrations in aqueous solutions were
determined by the quantitative method using multi elemental calibration solutions ICP-MS-68A and ICP-AM-6-A produced
by High-Purity Standards (Charleston, SC 29423, USA). Indium was used as an internal standard in all measurements. Information
detailing with the ICP-MS method used was presented in our previous publication [18].

Certified reference materials

For quality control, ten subsamples of the certified reference
materials (CRM) IAEA H-4 Animal muscle and IAEA HH-1
Human hair from the International Atomic Energy Agency (IAEA),
and also five sub-samples INCT-SBF-4 Soya Bean Flour, INCTTL-1
Tea Leaves and INCT-MPH-2 Mixed Polish Herbs from the Institute of Nuclear Chemistry and Technology (INCT, Warszawa,
Poland) were analyzed simultaneously with the investigated
prostate tissue samples. All samples of CRMs were treated in the
same way as the prostate samples. Detailed results of this quality
assurance program were presented in earlier publications [15,18].

Computer programs and statistic

A dedicated computer program for INAA mode optimization was
used [76]. All prostate samples for INAA-LLR were prepared in
duplicate and mean values of trace element contents were used in
final calculation. For elements investigated by both INAA-LLR
and ICP-MS methods the mean of all results was used. Using the
Microsoft Office Excel software Co/trace element contents for each
trace element in every sample were calculated. Then arithmetic
mean  standard error of mean was calculated for trace element
mass fraction and for ratios of Co/trace element mass fraction in
normal, benign hyperplastic and cancerous prostate. The difference
in the results between BPH and N, PCa and N, as well as PCa
and BPH was evaluated by parametric Student’s t-test and nonparametric
Wilcoxon-Mann-Whitney U-test. Values of p<0.05
were considered to be statistically significant. For the construction
of “individual data sets for Co/trace element mass fraction ratios
in normal, benign hypertrophic and cancerous prostate” diagrams
the Microsoft Office Excel software was also used.

Discussion

As was shown by us [14,15,17,18], the use of CRM IAEA H-4
Animal muscle, IAEA HH-1 Human hair, INCT-SBF-4 Soya
Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed
Polish Herbs as certified reference materials for the analysis of
samples of prostate tissue can be seen as quite acceptable. Good
agreement of the trace element contents in these CRMs, measured
by us using INAA-LLR and ICP-MS methods, with the certified
data [14,15,17,18] indicates an acceptable accuracy of the results
obtained in the present study

The mean values and standard error of mean (±SEM) were
calculated for 43 trace element contents including Co (Table 1),
as well as for 42 ratios of Co/trace element mass fractions (Table
2). The mass fraction of Co and other 42 trace elements were
measured in all, or a major portion of normal prostate samples.
The masses of BPH and PCa samples varied very strong from a
few milligrams (sample from needle biopsy material) to 100 mg
(sample from resected material). Therefore, in BPH and PCa
prostates mass fraction ratios of Co/Ag, Co/Cr, Co/Fe, Co/Hg,
Co/Rb, Co/Sb, Co/Sc, Co/Se, and Co/Zn were measured in all,
or a major portion of samples, while ratios to Co of other trace
element content were determined in 21 samples (11 BPH and 10
PCa samples, respectively).

Analysis of the mass fraction ratios for trace element in prostate
tissue could become a powerful diagnostic tool. To a large extent,
the resumption of the search for new methods for early diagnosis
of PCa was due to experience gained in a critical assessment of
the limited capacity of the prostate specific antigen (PSA) serum
test [77,78]. In addition to the PSA serum test and morphological
study of needle-biopsy cores of the prostate, the development
of other highly precise testing methods seems to be very useful.
Experimental conditions of the present study were approximated to the hospital conditions as closely as possible. In BPH and PCa
cases we analyzed a part of the material obtained from a puncture
transrectal biopsy of the indurated site in the prostate. Therefore,
our data allow us to evaluate adequately the importance of Co/
trace element mass fraction ratios for the diagnosis of PCa. As is
evident from Table 3 and, particularly, from individual data sets of
ratios (Fig. 1), the Co/Ag, Co/Al, Co/B, Co/Bi, Co/Li, Co/Mn, Co/
Pr, Co/Th, Co/Tl, and Co/Zr mass fraction ratios are potentially
the most informative test for a differential diagnosis. For example,
if 0.5 is the value of Co/Ag mass fraction ratio assumed to be the
upper limit for PCa (Fig. 1) and an estimation is made for “PCa or
intact and BPH tissue”, the following values are obtained:

Acknowledgements

We are grateful to Dr. Tatyana Sviridova, Medical Radiological
Research Center, Obninsk, and to the late Prof. A.A. Zhavoronkov,
Institute of Human Morphology, Russian Academy of Medical
Sciences, Moscow, for supplying prostate samples. We are also
grateful to Dr. Karandaschev V., Dr. Nosenko S., and Moskvina
I., Institute of Microelectronics Technology and High Purity
Materials, Chernogolovka, Russia, for their help in ICP-MS
analysis.
Competing interests
All other authors declare no competing interests.